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Page 1: A C. elegans large-scale genome-wide association study ... · A C. elegans large-scale genome-wide association study reveals hundreds of quantitative trait loci underlying responses

A C. elegans large-scale genome-wide association study reveals hundreds of quantitative trait loci underlying responses to biomedically relevant therapeutics

Genetic  Diversity  Toolkit

Abstract

Each red dot on the map above corresponds to the location where a wild C. elegans strain was isolated. To date we have 124 unique isotopes sequenced at approximately 84X coverage. This substantial level of coverage enables detection of genetic variation with high confidence. We use this genotypic variation in combination with phenotype data generated from our phenotyping platform to make phenotype-genotype associations.

Individuals in a population vary in a wide range of traits, including susceptibilities to diseases and responses to therapeutics. Identifying the genetic determinants of such traits remains difficult in human studies because of prohibitive genotyping costs, inability to control environmental conditions, and the polygenic nature of many traits. Caenorhabditis elegans provides a powerful model to probe the genetic determinants of these traits. Importantly, the molecular and genetic toolkits available in C. elegans allow us to characterize how genetic variation alters molecular mechanisms. We have optimized a high-throughput and high-accuracy phenotyping pipeline capable of quantifying various fitness traits of 96 genetically distinct strains exposed to 24 different environmental conditions in one week. Altogether, we exposed 96 wild isolates and 359 recombinant inbred lines to 70 different environmental perturbations, including chemotherapeutics, neuroactive compounds, anthelmintics, heavy metals, pesticides, and various temperatures and bacterial food sources. This approach identified more than 500 unique quantitative trait loci (QTL). As a proof of concept for our approach, we recapitulated a previously identified QTL that explains variation in response to the anthelmintic abamectin. Additionally, we identified five novel QTL that explain variation in response to abamectin treatment. We are actively pursuing another QTL, on the right arm of chromosome II that explains variation in response to the topoisomerase II poison etoposide. A scan of genetic variants underlying this QTL identified the candidate gene top-2, which encodes for one of the two known topoisomerase II proteins in the C. elegans genome. Strains sensitive to etoposide contain a top-2 gene with several predicted synonymous and non-synonymous variants when compared to the N2 genetic background. A top-2 deletion in the N2 genetic background failed to complement the sensitive top-2 variant phenotype, suggesting that top-2 is the causal gene underlying this QTL. We hypothesize that the Q797M variant present in sensitive strains leads to more stable binding of etoposide to TOPOII and therefore increases its potency as a poison. Our identification of conserved drug susceptibilities between humans and C. elegans and our ability to probe the genetic determinants of these susceptibilities has introduced C. elegans as a powerful model for elucidating the mechanisms underlying complex traits.

A) Manhattan plot showing that the right arm of chromosome II is associated with variation in animal size in response to the topoisomerase II inhibitor etoposide. B) Phenotypes of individual strains split by variants in the etoposide confidence interval. Five variants, which are in linkage disequilibrium with each other, in this confidence interval were most highly correlated with etoposide sensitivity and have the same phenotypic split (only one plot is shown). Four out of five are variants in the top-2 gene, which encodes for the C. elegans topoisomerase II. The fourth variant is in the neighboring gene npp-3, which encodes for a nucleoporin protein essential for nuclear pore formation. C) Linkage mapping data generated by phenotyping 350 recombinant lines generated from N2 and CB4856 parental lines. We identified an additional QTL on chromosome V using this reagent set. D) Linkage results showing that phenotypic variation in response to a second topoisomerase II poison, amsacrine, does not map to the right arm of chromosome II. This suggests that the peak on chromosome II is specific to etoposide treatment and will be discussed below.

Stefan Zdraljevic1,2, Sam K. Rosenberg1, Robyn E. Tanny1, Tyler C. Shimko1, and Erik C. Andersen1,2 1 Molecular Biosciences, Northwestern University, Evanston, IL, United States,2 Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, IL, United States

The above figure depicts all of the unique QTL identified in our screen. We quantified the phenotypes of 96 wild isolates in the presence of 70 environmental perturbations. Altogether, we identified 500 QTL. We manually verified that the QTL were not being driven by outliers. After manual curation of the phenotypes at each QTL, we ended up with 81 unique QTL, which are shown in the above figure.

Variation  in  top-­‐2    Leads  to  Differential  Etoposide  Sensitivity

A) Resistance to etoposide is dominant to sensitivity. B) We showed that the N2 ∆top-2 fails to complement the resistance of a natural allele in the CB4856 genetic background for body length reduction, indicating that top-2 is the causative gene for this phenotypic difference. An N2 npp-3 knockout strain complemented CB4856 sensitivity, suggesting that it is not the causal gene (not shown).

Summary  of  Identified  QTL

A) Multiple sequence alignment of the two C. elegans TOPOII alleles and the human TOPOII alpha and beta isoforms. The variant we hypothesize is contributing to phenotypic variability in response to etoposide in C. elegans also varies between the two human isoforms (red). There is debate in the community whether this residue is important for etoposide binding. B-C) Electron density of the topoisomerase II poison binding pocket in hTOPOII beta showing etoposide (B) and amsacrine (C) bound [1]. Wu et al. hypothesize that the methionine reside in the hTOPOII alpha leads to more stable binding of etoposide in the binding pocket, while other groups believe this residue is not important for binding because it is in two conformations in the hTOPOII beta crystal structure. We hypothesize that this residue is contributing to etoposide binding and stability and have a physiologically relevant way to quantify this effect. Our hypothesis is supported by variation in response to amsacrine treatment not mapping to the top-2 gene, suggesting to us that both N2 and CB4856 have normal topoisomerase II function. Additional support for this variant comes from the fact that the other highly correlated variants in the confidence interval are present in the hyper variable C-terminal domain of the protein. We have generated all the constructs to perform CRISPR/Cas9 mediated allele replacement to test our predictions.

Human TOPOIIα VKVAQLAGSVAEMSSYHHGEMSLMMTIINLAQNHuman TOPOIIß VKVAQLAGSVAEMSAYHHGEQALMMTIVNLAQNC.e. TOPOII R VKVAQLAGAVAEISAYHHGEQSLMGTIVNLAQDC.e. TOPOII S VKVAQLAGAVAEISAYHHGEMSLMGTIVNLAQD 780.......790.......800.......810

Future  Directions

Bro

od S

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IBiS

Funding

CMBD Training GrantIBiS Travel Award

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CB4856 N2 CB4856/∆top−2 N2/∆top−2

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CB4856 N2 N2N2−GFP

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N2−GFPN2−GFP

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Variant ReferenceGenotype

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Variant ReferenceGenotype

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I II III IV V X

0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20Genomic Position (Mb)

Treatment TypeAnthelmenticBacterial FoodChemotherapeuticHeavy MetalHerbicideNeuroactivePesticideTemperature

0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20Genomic Position (Mb)

Treatment TypeAnthelmenticBacterial FoodChemotherapeuticHeavy MetalHerbicideNeuroactivePesticideTemperature

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I II III IV V X

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LOD

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-log1

0(p)

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●●●●●●

●●●

0.0

2.5

5.0

7.5

10.0

0 5 10 150 5 10 15 0 5 10 0 5 10 15 0 5 10 15 20 0 5 10 15Genomic Position (Mb)

A B

C

D

A B

I II III IV V X45.99%

0

10

20

30

40

0 5 10 15 0 5 10 15 0 5 10 0 5 10 15 0 5 10 15 20 0 5 10 15Position (Mb)

LOD

scor

e

-log1

0(p)

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●●●●●●

●●●

0.0

2.5

5.0

7.5

10.0

0 5 10 150 5 10 15 0 5 10 0 5 10 15 0 5 10 15 20 0 5 10 15Genomic Position (Mb)

multiple hydrogen bonds with the side chains of N520 andE522 and the main-chain carbonyl group of R503. Incontrast, the arm in the major groove is more solvent-exposed and only a single hydrogen bond is presentbetween the drug and the protein. We noted that anearlier nuclear magnetic resonance-based study of the(protein-free) mitoxantrone-RNA binary complex placedboth alkylamino arms in the major groove (27); however,our modeling analysis indicates that a similar type ofbinding is not feasible in the context of the Top2 cleavagecomplex owing to steric conflicts between the drug andprotein residues in both DNA minor and major groove.Comparing the structures of Top2cc stabilized by differentdrugs clearly revealed that the binding of each drug in thecleavage site is mediated by a distinct and drug-specific setof interactions (Figure 5A–C).

Structure–activity relationships and the structural basisof drug resistance

Because the m-AMSA- and mitoxantrone-stabilizedTop2cc structures were obtained by post-crystallizationligand replacement rather than direct crystallization, thevalidity and pharmacological relevance of these new struc-tures must be carefully assessed. Toward this end, weexamined whether the structure–activity relationshipsand drug-resistant mutations known for each drug canbe fully accounted for by the observed interaction

patterns and drug-binding modes. Among the residuesinvolved in m-AMSA binding (Figure 3A), E522 appearsto play a key role with its side-chain carboxylate forming asalt bridge to the amine group of the 10-methanesulfonsubstitution, and its Cb-Cg-Cd moiety contacting the30-methoxy group and the C20 atom of the aniline ring(Figure 3A). This set of stabilizing interactions readilyexplains the impairment of drug activity on the removalof the 10- and 30-substitutions from the methanesulfon-m-anisidide head group (19), which is expected to substan-tially weaken drug binding. The m-AMSA-boundstructure also addresses why o-AMSA (Figure 1) is essen-tially inactive at inducing Top2-mediated DNA breakagecompared with the high potency exhibited by m-AMSA(26); steric clashes between the methoxy group and theE522 side chain would be produced when shifting themethoxy group from the meta (30, as in m-AMSA) tothe ortho position (20, as in o-AMSA). Therefore,despite the fact that the two AMSA stereoisomersdisplay comparable DNA-intercalating activity (26), o-AMSA and other AMSA analogs with ortho-substitutionsare incompatible with the minor groove drug-bindingpocket and are thus inefficient at stabilizing Top2cc (19).Intriguingly, when detached from the acridine chromo-phore, the methanesulfon-m-anisidide head group ofm-AMSA alone is capable of trapping Top2cc at highconcentrations (19). We suspect that by occupying theminor groove binding pocket, the anilino head group

Figure 5. Conformational landscapes of the drug-binding pockets for mammalian Top2-targeting anticancer drugs. (A–C) Surface/stick representa-tions of the etoposide-, m-AMSA- and mitoxantrone-binding sites, respectively. Selected DNA base pairs (purple) and protein (pink) residues areshown as surface representations and are labeled using white letters. Two hTop2bcore monomers are colored differently. Drugs (cyan) and the Y8210-conjugated+1 thymidines are shown as sticks. Labels belonging to the second monomer are flagged by a prime. Cyan letters designate polycyclic ringlabeling of drugs. (D and E) Superposition of residues 445–731 and residues 762–821 in hTop2b of the drug-stabilized hTop2cc structures to showstructural differences in the minor groove and major groove drug-binding pockets, respectively. Selected residues of hTop2cc structures stabilized byetoposide (blue), m-AMSA (yellow) and mitoxantrone (cyan) are shown as sticks. Side chain and main chain variations are indicated by red andblack arrows, respectively. Residues that exhibit no structural change under drug binding are labeled in gray.

10636 Nucleic Acids Research, 2013, Vol. 41, No. 22

at Northw

estern University Library on June 10, 2015

http://nar.oxfordjournals.org/D

ownloaded from

multiple hydrogen bonds with the side chains of N520 andE522 and the main-chain carbonyl group of R503. Incontrast, the arm in the major groove is more solvent-exposed and only a single hydrogen bond is presentbetween the drug and the protein. We noted that anearlier nuclear magnetic resonance-based study of the(protein-free) mitoxantrone-RNA binary complex placedboth alkylamino arms in the major groove (27); however,our modeling analysis indicates that a similar type ofbinding is not feasible in the context of the Top2 cleavagecomplex owing to steric conflicts between the drug andprotein residues in both DNA minor and major groove.Comparing the structures of Top2cc stabilized by differentdrugs clearly revealed that the binding of each drug in thecleavage site is mediated by a distinct and drug-specific setof interactions (Figure 5A–C).

Structure–activity relationships and the structural basisof drug resistance

Because the m-AMSA- and mitoxantrone-stabilizedTop2cc structures were obtained by post-crystallizationligand replacement rather than direct crystallization, thevalidity and pharmacological relevance of these new struc-tures must be carefully assessed. Toward this end, weexamined whether the structure–activity relationshipsand drug-resistant mutations known for each drug canbe fully accounted for by the observed interaction

patterns and drug-binding modes. Among the residuesinvolved in m-AMSA binding (Figure 3A), E522 appearsto play a key role with its side-chain carboxylate forming asalt bridge to the amine group of the 10-methanesulfonsubstitution, and its Cb-Cg-Cd moiety contacting the30-methoxy group and the C20 atom of the aniline ring(Figure 3A). This set of stabilizing interactions readilyexplains the impairment of drug activity on the removalof the 10- and 30-substitutions from the methanesulfon-m-anisidide head group (19), which is expected to substan-tially weaken drug binding. The m-AMSA-boundstructure also addresses why o-AMSA (Figure 1) is essen-tially inactive at inducing Top2-mediated DNA breakagecompared with the high potency exhibited by m-AMSA(26); steric clashes between the methoxy group and theE522 side chain would be produced when shifting themethoxy group from the meta (30, as in m-AMSA) tothe ortho position (20, as in o-AMSA). Therefore,despite the fact that the two AMSA stereoisomersdisplay comparable DNA-intercalating activity (26), o-AMSA and other AMSA analogs with ortho-substitutionsare incompatible with the minor groove drug-bindingpocket and are thus inefficient at stabilizing Top2cc (19).Intriguingly, when detached from the acridine chromo-phore, the methanesulfon-m-anisidide head group ofm-AMSA alone is capable of trapping Top2cc at highconcentrations (19). We suspect that by occupying theminor groove binding pocket, the anilino head group

Figure 5. Conformational landscapes of the drug-binding pockets for mammalian Top2-targeting anticancer drugs. (A–C) Surface/stick representa-tions of the etoposide-, m-AMSA- and mitoxantrone-binding sites, respectively. Selected DNA base pairs (purple) and protein (pink) residues areshown as surface representations and are labeled using white letters. Two hTop2bcore monomers are colored differently. Drugs (cyan) and the Y8210-conjugated+1 thymidines are shown as sticks. Labels belonging to the second monomer are flagged by a prime. Cyan letters designate polycyclic ringlabeling of drugs. (D and E) Superposition of residues 445–731 and residues 762–821 in hTop2b of the drug-stabilized hTop2cc structures to showstructural differences in the minor groove and major groove drug-binding pockets, respectively. Selected residues of hTop2cc structures stabilized byetoposide (blue), m-AMSA (yellow) and mitoxantrone (cyan) are shown as sticks. Side chain and main chain variations are indicated by red andblack arrows, respectively. Residues that exhibit no structural change under drug binding are labeled in gray.

10636 Nucleic Acids Research, 2013, Vol. 41, No. 22

at Northw

estern University Library on June 10, 2015

http://nar.oxfordjournals.org/D

ownloaded from

Chyuan-Chuan Wu et al. Nucleic Acids Research 2013 doi:10.1093/nar/gkt828

A B C